[Construction of CD138-targeted chimeric antigen receptor- modified T cells and their effect in multiple myeloma therapy].

Publisher: Chinese Medical Association Country of Publication: China NLM ID: 8212398 Publication Model: Print Cited Medium: Print ISSN: 0253-2727 (Print) Linking ISSN: 02532727 NLM ISO Abbreviation: Zhonghua Xue Ye Xue Za Zhi Subsets: MEDLINE

Publication: Beijing : Chinese Medical Association
Original Publication: Beijing : Zhongguo yi xue ke xue yuan.

Multiple Myeloma*/therapy ; Multiple Myeloma*/immunology ; Receptors, Chimeric Antigen*/immunology ; Syndecan-1*/immunology ; T-Lymphocytes*/immunology; Mice ; Animals ; Humans ; Hybridomas ; Immunotherapy, Adoptive/methods ; Cell Line, Tumor ; Receptors, Antigen, T-Cell/immunology ; Receptors, Antigen, T-Cell/genetics ; Antibodies, Monoclonal/immunology

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P <0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P <0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P <0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P <0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P <0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

81830005 National Natural Science Foundation of China; 2021-I2M-1-041 CAMS Innovation Fund for Medical Sciences

Keywords: Antigen recognition domain; CD138; Chimeric antigen receptor; Immunotherapy; Multiple myeloma
Local Abstract: [Publisher, Chinese] 目的： 构建一种新型靶向CD138的嵌合抗原受体T（CAR-T）细胞，探索其抗恶性浆细胞肿瘤作用。 方法： 通过单克隆抗体制备及筛选技术，获得能稳定分泌CD138抗体的杂交瘤细胞株；将杂交瘤细胞接种至小鼠腹腔，收集腹水并纯化得到抗CD138抗体纯品，进一步检测抗体特异性及亲和力；RT-PCR扩增其可变区序列，以此作为抗原识别域构建CD138 CAR，并表达于T细胞表面，制备CD138 CAR-T；流式细胞术检测CAR-T细胞表型特征；体外杀伤及脱颗粒实验检测其抗肿瘤作用。 结果： ①成功制备抗人CD138抗体杂交瘤细胞株，并筛选获得稳定分泌抗人CD138抗体的杂交瘤细胞株5G2。②CD138（5G2）抗体可以特异性识别CD138(+)细胞，与CD138蛋白亲和常数（K(D)）为6.011×10(-9) mol/L，与CD138(-)细胞无明显交叉反应。③应用分子克隆技术扩增得到CD138（5G2）抗体可变区序列，成功构建CD138（5G2）CAR慢病毒载体，通过感染T细胞获得的CD138（5G2）CAR-T细胞，可以有效结合人CD138重组蛋白。④CD138（5G2）CAR-T可以有效大量扩增，表型检测发现CD138（5G2）CAR-T细胞更多的向中心记忆T及记忆干细胞方向分化，终末分化效应T细胞比例降低。⑤与靶细胞共培养48 h后，与Vector-T细胞相比，CD138（5G2）CAR-T细胞可以有效杀伤CD138(+)骨髓瘤细胞系H929[效靶比为1∶2，（12.92±8.02）%对（54.25±15.79）%， P <0.001]。但对CD138(-) K562细胞系无明显杀伤作用。⑥脱颗粒实验显示，H929细胞可以显著激活CD138（5G2） CAR-T细胞，但对Vector-T细胞无明显激活作用[（25.78±3.35）%对（6.13±1.30）%， P <0.001]。⑦与CD138(+)细胞共培养后CD138（5G2）CAR-T分泌细胞因子水平较Vector-T组明显升高[IL-2：（1 697.52±599.05）pg/ml对（5.07±1.17）pg/ml， P <0.001；IFN-γ：（3 312.20±486.38）pg/ml对（9.28±1.46） pg/ml， P <0.001；TNF-α：（1 837.43±640.49）pg/ml对（8.75±1.65）pg/ml， P <0.001]，但与CD138(-)细胞共培养后两组间细胞因子分泌水平无明显差异。 结论： 本研究成功制备了抗CD138单克隆抗体，以其抗原识别域构建的CAR-T细胞可以有效发挥抗肿瘤作用，为人CD138抗原的检测及多发性骨髓瘤的免疫治疗提供新的选择。.

0 (Receptors, Chimeric Antigen)
0 (Syndecan-1)
0 (SDC1 protein, human)
0 (Receptors, Antigen, T-Cell)
0 (Antibodies, Monoclonal)

Date Created: 20240704 Date Completed: 20240704 Latest Revision: 20240730

20240730

10.3760/cma.j.cn121090-20240131-00047

38964917